A. Technical Field
The use of microbial inoculants to promote plant health is known. Generally, microbes, including bacteria and fungi, may be applied to a plant to improve plant nutrition, promote plant growth, provide resistance to disease and to treat disease. Examples of microbial inoculants include plant growth promoting rhizobacteria such as Rhizobium sp. which increase nitrogen nutrition in leguminous crops such as soybean and chickpeas, phosphate-solubilising bacteria such as Agrobacterium radiobacter, fungal inoculants including mycorrhizal fungi and endophytic fungi, such as Piriformis indica, which provide plant nutrition benefits, and composite inoculants which have shown synergistic effects on plant growth and nutrition.
An endophyte is an endosymbiont, often a bacterium or fungus, that lives within a plant for at least part of its life without causing apparent disease. Endophytes are ubiquitous and have been found in all the species of plants studied to date. Endophytes may be transmitted either vertically (directly from parent to offspring) or horizontally (from individual to unrelated individual). Vertically transmitted fungal endophytes are typically asexual and transmit from the maternal plant to offspring via fungal hyphae penetrating the host's seeds. Since their reproductive fitness is intimately tied to that of their host plant, these fungi are often mutualistic. Conversely, horizontally transmitted fungal endophytes are sexual and transmit via spores that can be spread by wind and/or insect vectors. Endophytes can benefit host plants by preventing pathogenic organisms from colonizing them. Extensive colonization of the plant tissue by endophytes creates a “barrier effect,” where the local endophytes out compete and prevent pathogenic organisms from taking hold. Endophytes may also produce chemicals which inhibit the growth of competitors, including pathogenic organisms.
Various endophytes, particularly fungi, have been used in order to manage plant diseases by targeting the growth and viability of plant pathogens. In addition to their diverse utility, microbial inoculants can replace or significantly reduce the need to use harmful chemical fertilizers and pesticide treatments, which is becoming more important as regulations imposing stringent restrictions on the use of such chemicals come into force. The use of biocontrol and biostimulant fungal organisms in conventional field and horticultural crops is still relatively new. Published research has covered the use of many fungal organisms as aids in agriculture.
Clonostachys rosea (previously known as Gliocladium roseum) is recognized as a beneficial organism. Clonostachys rosea is a species of fungus in the family Bionectriaceae that colonizes living plants as an endophyte. Clonostachys rosea must be able to establish either endophytically in, or epiphytically on, plant organs, but the latter is not significant in the field (except perhaps in cases of roots), because the organism is significantly controlled by UV-A and UV-B. The use of Clonostachys rosea endophytes is preferable to some other biological control agents, because Clonostachys rosea are rapid internal colonizers, with better ability to compete against other organisms. There are a variety of Clonostachys rosea strains, which all share the same common features of growing quickly, having a felt-like mycelium, and having no detrimental effects on higher plants.
Clonostachys rosea is a locally systemic endophyte often termed translaminar, i.e. it moves from top of leaf to bottom colonizing tissue throughout the sprayed/inoculated area. Generally, there is no movement into stem or leaves from roots. A spray on flower parts may colonize seed. Clonostachys rosea has no sexual stage, and conidia is spread from cotyledons of soy or from inside leaves of rose that are digested and form conidia.
The modes of action of C. rosea as a biological control agent are not fully known, although site occupation, mycoparasitism, competition for nutrients, and secondary metabolite production have been suggested to play significant roles. The lack of understanding of the role of Clonostachys rosea and other endophytes in the life of plants is often dominated by the misconception that toxins or antibiotics are always involved, and the classification of endophytic organisms as biopesticides often missed the actual role in plant health.
The mode of action of Clonostachys rosea strains involves colonization and possession of a root system or treated portion of a plant by mycelium, followed by denial of food to other organisms. In microbial interaction, possession is 9/10ths of the law. Early colonization of seed, foliage, flowers and fruit achieves the best results.
Additionally, current research has postulated that C. rosea secrete hydrophobins, which are small proteins produced only by filamentous fungi, which forms amphipathic layers on the outer surface of fungal cell walls. (Dubey et al, Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea, BMC Microbiology 2014, 14:18). The hydrophobic side of the amphipathic layer is exposed to the outside environment, while the hydrophilic side is directed towards cell wall polysaccharides. It has been reported that Clonostachys rosea can secret subtilisin-like extracellular serine proteases or potentially other substances during the infection. C. rosea produces the enzyme zearalenone hydrolase (ZHD101), which degrades Zearalenone (“ZEA”), which is produced by mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum. A mycotoxin that exhibits antifungal growth. Zealerones are mycotoxins with estrogenic-mimic activity. (Kakeya, Biotransformation of the Mycotoxin, Zealerone, to a Non-estrogenic Compound by a Fungal Strain of Clonostachys sp., Bioscience, Biotechnology, and Biochemistry, Vol. 66 (2002) No. 12 P 2723-2726). In one case, it has been reported that pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes entry into the nematode body and subsequent death and degradation of the nematodes. (Zhang et al, Investigation on the infection mechanism of the fungus Clonostachys rosea against nematodes using the green fluorescent protein, Applied Microbiology and Biotechnology April 2008, Volume 78, Issue 6, pp 983-990).
B. Description of Related Art
Clonostachys rosea strains tolerate certain fungicides, and thus Clonostachys rosea has been considered a candidate for integrated pest management system.
Bio-priming of seeds has been well-known. See, for example, Callan, Bio-priming Seed Treatment for Biological Control of Pythium ultimum Preemergence Damping-off in sh2 Sweet Corn, Plant Disease, Vol. 74 No. 5 (1990); Rao, Bio-Priming Of Seeds: A Potential Tool In The Integrated Management Of Alternaria Blight Of Sunflower, HELIA, 32, Nr. 50, p.p. 107-114, (2009);
The published literature indicates that seeds are subjected to fungicidal treatment, followed by inoculation of a biological organism. Thus, Rao, 2009, describes that for integrated seed treatment options tested for the management of Alternaria blight of sunflower, the highest benefit was obtained in the seed treatment with Carbendazim+Iprodione (Quintal) at 0.3% in water along with hexaconazole foliar spray (0.1%) followed by seed treatment with Pseudomonas fluorescens (0.8%) in jelly+hexaconazole foliar spray.
The published literature for crops indicates that it has been generally considered that Clonostachys rosea is not applied together with fungicides. See for example, U.S. Pat. No. 6,495,133 to Xue who reported that “ACM941 plus 50% of the regular rate of thiram was the most effective treatment, which increased yield by 21% . . . . The results also indicated that ACM941 bioagent is compatible with thiram fungicide,” and that “Results of this study also indicated that ACM941 bioagent is compatible with metalaxyl fungicide. Subsequent research by Sutton and Brown has shown that thiram and not metalaxyl is toxic to Clonostachys rosea ACM941. An enhanced effectiveness was generally observed when ACM941 was combined with metalaxyl fungicide.” See also Macedo et al, Sensitivity of four isolates of Clonostachys rosea to pesticides used in the strawberry crop in Brazil, J. Pestic. Sci. 37(4), 333-337 (2012). Macedo et al determined the sensitivity of four isolates of Clonostachys rosea to fungicides and other pesticides, and concluded that all fungicides inhibited mycelial growth and conidia germination of all isolates. For that reason, research in the area has generally compared Clonostachys rosea to fungicides in side by side tests, rather than in an integrated manner. See, for example, Xue et al. Biological control of Fusarium head blight of wheat with Clonostachys rosea strain ACM941, Can. J. Plant Pathol. 31: 169-179 (2009).
An inoculant composition comprising Clonostachys rosea is disclosed in United States Patent Publication 20120021906A1 to Sutton and Mason. Sutton and Mason disclose a composition of a carrier having a moisture content of not more than about 5%, and a method of inoculating a plant to promote growth, enhance resistance to adverse conditions or promote re-growth is also provided comprising applying the inoculant composition to the plant.